Virulent orthopoxviruses (OPV) such as monkeypox and smallpox represent Category A select agents that could be used as biological weapons. Although smallpox is now extinct in nature, it was weaponized by the former Soviet Union and remains a significant concern due to the potential that remaining stocks of this highly contagious virus could fall into the hands of terrorists or rogue states. Monkeypox is less contagious, but induces disease manifestations similar to discrete smallpox and results in ~10% mortality in unvaccinated populations. For these reasons, monkeypox can easily be mistaken for smallpox and may lead to widespread panic during a bioterrorist attack. Monkeypox is still endemic in regions of the Democratic Republic of Congo (DRC) and possibly other parts of Africa. In 2003, monkeypox spread beyond the borders of the African continent and resulted in a substantial outbreak in the United States involving at least 72 potential or confirmed cases. Monkeypox is easily obtained, is stable when delivered by aerosol, and has the potential to be genetically manipulated for increased virulence. Therefore both monkeypox and smallpox represent a significant threat to homeland security in the United States. In this application, our goal is to develop rapid, point-of-care diagnostic tests capable of diagnosing disseminated orthopoxvirus infections such as monkeypox (as well as smallpox through a pan-orthopoxvirus detection system) in the context of true outbreak conditions. Chickenpox (varicella zoster virus, VZV) is commonly mistaken for disseminated orthopoxvirus infection and for this reason we will also develop rapid VZV-specific tests to use for differential diagnosis. Using an integrated dual-detection system, these novel diagnostic tests will combine serological analysis with direct detection of the pathogen to provide the broadest window of detection for identifying orthopoxvirus outbreaks. [unreadable] The Specific Aims of this application include: [unreadable] 1) Rapid serological diagnosis of OPV infection [unreadable] 2) Differential serological diagnosis of OPV versus VZV infection [unreadable] 3) Direct detection of OPV and VZV viral antigens in clinical specimens [unreadable] [unreadable] It is feared that viruses such as smallpox (30% mortality rate) or monkeypox (10% mortality rate) will be used as biological weapons. To aid in biodefense against these viruses, we plan to develop effective dual-detection diagnostic assays that allow rapid identification of these pathogens or the immune response to these pathogens. We plan to concentrate our studies on human monkeypox, itself a potential bioterrorism agent, because it also provides the most appropriate model for allowing real-life assessment of diagnostic capabilities during human outbreak situations and most closely mimics human smallpox which is now extinct in nature. [unreadable] [unreadable] [unreadable]